The goal of this Project is to understand how virus infection abrogates peripheral transplantation tolerance in naive mice and why pre-existing virus-immunity appears to compromise transplantation tolerance induction. During the previous funding period, we documented that graft durability after peripheral transplantation tolerance induction is adversely affected by virus infection in a time-dependent manner. 1) Graft survival in mice treated with costimulation blockade is shortened if the recipients are LCMV-immune. 2) Infection at the time of tolerance induction prevents graft survival, probably by preventing the deletion of alloreactive CD8 + T cells. 3) Virus infection after the deletion of alloreactive CD8 + T cells is less deleterious but still shortens graft survival. 4) Virus infection of hosts with healed in allografts, however, does not shorten graft survival. We also documented in mice that viral infection during peripheral tolerance induction can compromise recipient survival. To understand the mechanisms underlying the complex interrelationships of transplantation tolerance induction and virus infection, we propose three Specific Aims. Specific Aim 1 will test the hypothesis that virus infection will activate and mature dendritic cells to provide bystander activation of alloreactive T cells and prevent their deletion by costimulation blockade. Specific Aim 2 will test the hypothesis that virus infection prevents the generation of regulatory CD4+CD25 + T cells by costimulation blockade Specific Aim No. 3 will test the hypothesis that virus-induced allo-cross-reactive memory T cells shorten allograft survival in mice treated with costimulation blockade. To test these hypotheses, we will use two recently developed innovative technologies. For studying viral immunity, we will use techniques developed in the third project and the Virology Core for identifying virus-immune T cells and determining their antigen-specificity at the single cell level. For studies of transplantation, we will use a newly developed "synchimera" model based on TCR transgenic mice to quantify alloreactive T cells. Our data will permit the mechanisms underlying the relationship between viral infection and tolerance induction to be determined in advance of the introduction of tolerance-based transplantation clinical trials.